The present invention relates to a novel ligand polypeptide for the G protein-coupled receptor protein, APJ and a DNA comprising a DNA encoding the ligand polypeptide.
Many hormones and neurotransmitters mediate biological functions through specific receptors present on the cell membrane. Many of these receptors engage themselves in the intracellular transduction of signals through activation of the coupled guanine nucleotide-binding protein (hereinafter sometimes referred to briefly as G protein) and have the common structure comprising 7 transmembrane domains. Therefore, these receptors are collectively referred to as G protein-coupled receptor or 7-transmembrane receptor.
Through the interaction of such hormones or neurotransmitters with G protein-coupled receptors, a variety of regulatory functions of vital importance, for example maintenance of homeostasis, reproduction, development, metabolism and growth, and regulation of the nervous system, circulatory system, immune system, gastrointestinal system and metabolic system are all discharged. While it is known, as mentioned above, that receptor proteins exist for various hormones or neurotransmitters and are playing important roles in the control of vital functions, it is still unknown in many respects whether unknown active substances (hormones or neurotransmitters) and their receptors ever exist or not.
In recent years, by taking advantage of the similarity of such G protein-coupled receptor proteins in partial amino acid sequence, searches for DNAs coding for novel receptor proteins have been undertaken by the polymerase chain reaction technique (abbreviated as PCR) and actually a large number of orphan G protein-coupled receptors, so called because their ligands are unknown, have been cloned to this day (Libert, F., et al., Science, 244, 569-572, 1989; Welch, S. K., et al., Biochem. Biophys. Res. Commun., 209, 606-613, 1995; Marchese, A., et al., Genomics, 23, 609-618, 1994; Marchese, A., Genomics, 29, 335-344, 1995). Furthermore, as the result of random sequencing of genomic DNAs and cDNAs, novel G protein-coupled receptor proteins have been discovered one after another (Nomura, N., et al., DNA Research, vol. 1, 27-35, 1994). The only general means so far available for determining the ligands to such orphan G protein-coupled receptor proteins is a mere estimation based on the similarity in primary structure among G protein-coupled receptor proteins. However, many orphan G protein-coupled receptor proteins have low homology with known receptors and it is, therefore, difficult to estimate their ligands based on the similarity in primary structure alone except in those cases where they are actually subtypes of receptors for known ligands. On the other hand, since many orphan G protein-coupled receptors have been discovered as the result of gene analysis, it is supposed that there still exist many unknown corresponding ligands. However, only in a few cases, ligands have actually been identified for such orphan G protein-coupled receptors.
Recently, investigation for novel opioid peptides by introducing a cDNA coding for a receptor protein which a ligand is unknown, i.e. an orphan G protein-coupled receptor protein, into animal cells have been reported (Reinsheid, R. K. et al., Science, 270, 792-794, 1995, Menular, J. -C., et al., Nature 377, 532-535, 1995). However, in view of similarities to known G protein-coupled receptor proteins and tissue distributions, it could be easily anticipated in these cases that the ligand would be belonging to the family of opioid peptides. The history of research and development in the realm of substances acting on the living body through the opioid receptor dates back to many years ago and various antagonists and agonists have been developed. Therefore, among the compounds artificially synthesized, an agonist of the receptor was picked out and, using it as a probe, expression of the receptor in the receptor cDNA-transfected cells was verified. Then, a search was made for an activator of the intracellular signal transduction which was similar to the agonist, the activator so found was purified, and the structure of the ligand was determined. However, when the homology of an orphan receptor to known G protein-coupled receptor proteins is low, it was very difficult to predict its ligand.
APJ is one of the orphan G protein-coupled receptors so far reported (O""Dowd, B. F., et al., Gene, 436, 355-359, 1993). APJ has low homology with an angiotensin receptor (AT1) but its ligand has remained unknown since no reaction was detected at all with angiotensin II despite the expression of APJ in CHO cells.
Ligands for APJ, which is an orphan G protein-coupled receptor expressed in the central nervous system, circulatory system, reproductive system, immune system, digestive organs and so forth, are expected to be useful as drugs but their structure and functions have not been elucidated as yet.
Employing a cell in which a cDNA coding for orphan G protein-coupled receptor protein (APJ) has been expressed by a suitable means and using measurement of a specific cell stimulation activity exemplified by a signal transduction activity as an indicator, the inventors of the present invention succeeded in screening for a polypeptide which said receptor protein recognizes as a ligand.
Furthermore, the inventors found that a compound can be screened which is capable of changing the binding activity of this ligand which is an activating factor to said receptor protein.
The present invention, therefore, relates to
(1) a polypeptide capable of binding to a receptor protein which comprises an amino acid sequence represented by SEQ ID NO:3 or a substantial equivalent thereto, a precursor polypeptide thereof, its amide or ester, or a salt thereof,
(2) a polypeptide as mentioned in the above (1), which comprises an amino acid sequence represented by SEQ ID NO:1 or a substantial equivalent thereto,
(3) a polypeptide as mentioned in the above (1), which comprises a partial sequence of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(4) a polypeptide as mentioned in the above item (1), which comprises
(a) a peptide comprising the 6th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(b) a peptide comprising the 40th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(c) a peptide comprising the 42nd to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(d) a peptide comprising the 47th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(e) a peptide comprising the 61st to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(f) a peptide comprising the 65th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42, or a derivative thereof resulting from conversion of the N-terminal amino acid (Gln) to a pyroglutamic acid residue,
(g) a peptide comprising the 1st to 25th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(h) a peptide comprising the 6th to 25th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(i) a peptide comprising the 42nd to 64th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(j) a peptide comprising the 61st to 64th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(k) a peptide comprising the 43rd to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(l) a peptide comprising the 41st to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(m) a peptide comprising the 66th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(n) a peptide comprising the 67th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(o) a peptide comprising the 64th to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(p) a peptide comprising the 63rd to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(q) a peptide comprising the 65th to 76th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(r) a peptide comprising the 65th to 75th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42, or
(s) a peptide comprising the 65th to 75th amino acid residues of the amino acid sequence represented by SEQ ID NO:15, 38, 40 or 42,
(5) a polypeptide as mentioned in the above (1) which comprises an amino acid sequence of from the 65th amino acid residue to the 77th amino acid residue of the amino acid sequence represented by SEQ ID NO:15, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42,
(6) a polypeptide as mentioned in the above (1) which has the amino acid sequence: pGlu Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe,
(7) a polypeptide as mentioned in the above (1) which comprises an amino acid sequence of from the 42nd amino acid residue to the 77th amino acid residue of the amino acid sequence represented by SEQ ID NO:15, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42,
(8) a polypeptide as mentioned in the above (1) which is a precursor polypeptide having an amino acid sequence represented by SEQ ID NO:15 or a substantial equivalent thereto,
(9) a polypeptide as mentioned in the above (1) which is a precursor polypeptide having an amino acid sequence represented by SEQ ID NO:38 or a substantial equivalent thereto,
(10) a polypeptide as mentioned in the above (1) which is a precursor polypeptide having an amino acid sequence represented by SEQ ID NO:40 or a substantial equivalent thereto,
(11) a polypeptide as mentioned in the above (1) which is a precursor polypeptide having an amino acid sequence represented by SEQ ID NO:42 or a substantial equivalent thereto,
(12) a DNA which comprises a DNA having a nucleotide sequence coding for a polypeptide capable of binding to a receptor protein which comprises an amino acid sequence represented by SEQ ID NO:3 or a substantial equivalent thereto, or a precursor polypeptide thereof,
(13) a DNA as mentioned in the above (12) wherein the polypeptide encoded thereby comprises an amino acid sequence represented by SEQ ID NO:1 or a substantial equivalent thereto,
(14) a DNA as mentioned in the above (12) wherein the polypeptide encoded thereby comprises an amino acid sequence of from the 65th amino acid residue to the 77th amino acid residue of the amino acid sequence represented by SEQ ID NO:15, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42,
(15) a DNA as mentioned in the above (12) wherein the polypeptide encoded thereby comprises an amino acid sequence of from the 42nd amino acid residue to the 77th amino acid residue of the amino acid sequence represented by SEQ ID NO:l5, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42,
(16) a DNA as mentioned in the above (12) wherein the precursor polypeptide encoded thereby comprises an amino acid sequence represented by SEQ ID NO:15 or a substantial equivalent thereto,
(17) a DNA as mentioned in the above (12) wherein the precursor polypeptide encoded thereby comprises an amino acid sequence represented by SEQ ID NO:38 or a substantial equivalent thereto,
(18) a DNA as mentioned in the above (12) wherein the precursor polypeptide encoded thereby comprises an amino acid sequence represented by SEQ ID NO:40 or a substantial equivalent thereto,
(19) a DNA as mentioned in the above (12) wherein the precursor polypeptide encoded thereby comprises an amino acid sequence represented by SEQ ID NO:42 or a substantial equivalent thereto,
(20) a recombinant vector which comprises the DNA as mentioned in the above (12),
(21) a transformant carrying the DNA as mentioned in the above (12) or the recombinant vector as mentioned in the above (20),
(22) a method for producing a polypeptide, a precursor polypeptide thereof or a salt thereof which comprises cultivating the transformant as mentioned in the above (21),
(23) a pharmaceutical composition which comprises the polypeptide, the precursor polypeptide thereof, its amide or ester, or a salt thereof as mentioned in the above (1),
(24) a pharmaceutical composition which comprises the DNA as mentioned in the above (12),
(25) a pharmaceutical composition as mentioned in the above (23) or (24) which is a central nervous system function modulator, a circulatory function modulator, an immune function modulator, a gastrointestinal function modulator, a metabolic function modulator or a reproductive function modulator,
(26) an antibody against the polypeptide or the precursor polypeptide thereof as mentioned in the above (1),
(27) a diagonostic composition which comprises the antibody as mentioned in the above (26), and
(28) a method for screening for a compound changing binding activity of the polypeptide as mentioned in the above (1) to a receptor protein which comprises an amino acid sequence represented by SEQ ID NO:3, which comprises using the polypeptide as mentioned in the above (1) and said receptor protein,
The present invention further provides:
(29) a polypeptide as mentioned in the above (1) which is of mammalian origin, and
(30) a pharmaceutical composition as mentioned in the above (23) or (24) which is an agent for the treatment and/or prevention of dementia, depression, hyperactive child syndrome (microencephalopathy), disturbance of consciousness, anxiety disorder, schizophrenia, phobia, growth hormone secretory disorder, hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia, hyperlipemia, hyperprolactinemia, diabetes mellitus, cancer, pancreatitis, diseases of kidney, Turner""s syndrome, neurosis, rheumatoid arthritis, spinal injury, transient brain ischemia, amyotrophic lateral sclerosis, acute myocardial infarction, spinocerebellar degeneration, bone fracture, wounds, atopic dermatitis, osteoporosis, asthma, epilepsy, sterility, arteriosclerosis, pulmonary emphysema, pulmonary edema, galactorrhea, AIDS, or the like.
In the practice of the present invention, the G protein-coupled receptor protein for the ligand polypeptide is specifically:
(31) a G protein-coupled receptor protein characterized by containing an amino acid sequence represented by SEQ ID NO:3, or a substantial equivalent thereto, or a salt thereof, or
(32) a G protein-coupled receptor protein as mentioned in the above (31) which comprises an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:3 by deletion of 1 to 30, preferably 1 to 10 amino acid residues therefrom, by addition (or insertion) of 1 to 30, preferably 1 to 10 amino acid residues thereto (or thereinto), or by substitution of 1 to 30, preferably 1 to 10 amino acid residues with a different amino acid residue or residues, or a salt thereof, for instance.
In the present specification, the term xe2x80x9csubstantial equivalent(s)xe2x80x9d means that the activity of the protein, e.g., nature of the binding activity of the ligand and the receptor and physical characteristics are substantially the same. Substitutions, deletions or insertions of amino acids often do not produce radical changes in the physical and chemical characteristics of a polypeptide, in which case polypeptides containing the substitution, deletion, or insertion would be considered to be substantially equivalent to polypeptides lacking the substitution, deletion, or insertion. Substantially equivalent substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.